Ultrasonographic sex determination of the flathead grey mullet (Mugil cephalus)

Abstract Background Determination of sex in fish without external sexual dimorphism is important. Sex determination of the flathead grey mullet (Mugil cephalus) in the culture system and spawn‐producing and fish reproduction system is very important. Currently, there are several methods to determine the sex of fish; however, they are time‐consuming, expensive and sometimes dangerous for the fish. Objective The purpose of this study was the sex determination of flathead grey mullet by ultrasonography as a safe and accurate method. Methods In this regard, 30 flathead grey mullets (early in their maturation) from a farm in Gomishan‐Golestan province, Iran were examined using a portable ultrasound machine with a 6–12 MHz waterproof linear transducer. Scanning was performed on the ventral body surface in the sagittal and transverse planes, between ventral and anal fins. Results Differences in shape, qualitative size, echogenicity and the wall layers of the ovary and testis were evaluated for sex determination. Results of ultrasonography were confirmed immediately at necropsy. Ultrasonographic accuracy for sex determination was 100%. The speed of the procedure was around 30 s per fish. Conclusions Overall, the sex determination of flathead grey mullet by ultrasonography was high in accuracy and speed, non‐invasive and safe for fish and consumers, and may be considered a priority. Place of the organs in the M. cephalus coelomic cavity and the obtained experience showed that for rapid and easy sex identification, the best place to put the transducer is on the ventral surface of the fish in the caudal part of the coelomic cavity for taking transverse and sagittal ultrasonograms.


INTRODUCTION
Flathead grey mullet which is grey or striped are members of the family of Mugilidae. Mugilidae are among food fish and often are farmed due to the fact that they grow fast (Aoki et al., 2011).
Stripped Mugilidae is found in coastal waters of the tropical, semitropical and temperate areas in seawater, salty and also freshwater.
This kind is one of farmed sea fish all over the world. These fishes have an appropriate situation for farming: great resistance against a vast scope of temperature and salinity; good growth coefficient; appropriate food changing coefficient; excellent public approval; the possibility to form polyculture with shrimp, cream fish and even carp fish as one of the best kinds of farmed sea fish in various parts of the world like Italy, countries located in northern Africa (like Egypt), occupied Palestine, India, Pakistan, Japan, Hong Kong, Taiwan, Vietnam, Indonesia and the coastal countries on the coast of the southern Pacific Ocean and Hawaii (Kuo & Nash, 1975 Mugilidae was met with failure (Kuo & Nash, 1975). Because the sperm of this kind of fish is activated in the salinity 14 in 1000 and in the salinity lower than 13-14 in 1000 in the Caspian Sea, this kind could not reproduce and become extinct.
The artificial culture of Mugilidae was carried out for the first time in 1930 in Italy with a technique similar to modern salmon farming.
In Iran, in order to reproduce grey Mugilidae artificially from agricultural land farmed productively, small fish imported from Hong Kong were farmed in soil pool stations of fish farming in the salty water of Gomishan-Golestan province, Iran were used.
Because in these fish no physical change occurs before maturation, the differentiation between the sexes of striped Mugilidae is of particular importance management of producing productive fish for the activities of hatcheries and artificial culture. Artificial culture in this species after hormone stimulation, spawning and powering sperm is done naturally and by observing the 2-1 ratio of male to female. On the other hand, in nature, the lack of males in the herd is a natural issue, and it is this lack of males which doubles the importance of sex identification in hatcheries. In growing farms, the sex distinction brings carriers of this exported species to European countries and the USA, the majority of these farmers in Hong Kong, Taiwan and other such countries have made efforts to farm mono sex culture (Chang et al., 1999).
There are different methods to determine sex in the fishes. One of which is sex determination through the sexual hormones from the blood. However, taking a sample from the blood confers great stress for the fish and in the experts' point of view determining the amount of hormones in the blood is difficult to perform because of the need for advanced devices to evaluate the amount of sex hormones, which is time-consuming and costly .
Other techniques are using trocar or operation cutting on the ventral wall which is aggressive, especially in the case of Mugilidae which are sensitive fish (Chang et al., 1999).
Using these aggressive methods can lead to the death of the fish. In techniques like using a trocar or cutting the body of the fish, because the area under examination has restricted length and the length of gonads is not examined, there is a possibility that in the cut body, the gonads are not observed or the gonads have not yet undertaken changes that would allow us to distinguish the male and the female.
Using ultrasonography for sex identification in the fish is a new technique that dates back to the years after 1980. For the first time, Martin et al. in 1983 used ultrasonography to identify sexes in 20 live fish (Coho salmon) (Martin & Rommens, 1983). After that, Reimers et al. in 1987 used it in Rainbow trout and the Atlantic salmon (Reimers et al., 1987), and several other types of research studies were conducted in this field until 1993 when Reamers et al. used ultrasound as part of a technique to identify sexual maturity in farmed Atlantic salmon and expressed hope this technique could reform fish artificial breeding and gain more benefits for the farmers (Reimers et al., 1993).
Vajhi used ultrasonography in sex identification on Acipenser gueldenstaedtii persicus and after that on Acipenser stellatus (Moghim et al., 2002;Vajhi et al., 1999). These researchers stated that the accuracy of sex identification is totally 97.2% (Moghim et al., 2002). Vajhi stated while continuing their research studies in 2003 on sturgeon (Acipenser nudiventris), that the identification in young males is difficult (Vajhi et al., 2003). In another research which was conducted by Colombo et al. (2004), the accuracy of this technique was stated to be 86%. Finally, in the research studies by Masoudifard in 2011, ultra-sonography was used to identify sex in immature Beluga. They stated this technique can differentiate males from females in 3-year-old fish .
The place to put the transducer on the body of the fish is of special importance. In a way that in the Salmo salar (Mattson, 1991), Gadus morhua (Karlson & Holm, 1994), Acipenser persicus (Vajhi et al., 1999) and A. stellatus (Moghim et al., 2002), the fish was laid backwards for ultrasonography, and the transducer was put on the ventral surface of the body between pectoral and anal fins (caudal part of the coelomic cavity). In 3-year-old beluga (  for anatomical study in live animals (Zehtabvar et al., 2018). Ultrasonography is also a very suitable method for the anatomical study of various structures (Goddard, 1995).

Individuals
In order to conduct this research, we went to Gomishan in Golestan province in Iran, and 30 M. cephalus (4-year old, early in their maturation) were studied. Information on the samples is presented in Table 1.
All the fish were kept in a pond. Fishes were taken from different parts of the pool for this study. In addition, 10 fish that had already been caught and refrigerated were used for a preliminary anatomical study before ultrasonography (among the fishes caught for sale and food).

Anatomical study
Before ultrasonography, the topographic and morphologic examination was conducted on existing fish, and the fish anatomy information resulted, then early familiarization with ultrasonograms of male gonads and female gonads regarding their shape scope and echogenicity, the ultrasonography of this organ was done out of the body in water. After that, the fish anatomy was performed, and sex determination based on the morphology of gonads was carried out and recorded. For this step, 10 fish that have already been caught and kept in the refrigerator were used (mentioned in Section 2.1).

Ultrasonographic study
In order to determine the sex of samples, a portable SonoSite- In order for the ultrasonography of gonads inside the body, it was necessary for the fish to be anaesthetized. In order to do that, we used TMS (MS-222), (3-aminobenzoic acid ethyl ester methanesulfonate) (1.25 g/ml water). Then the fish was taken to a tub full of water, and ultrasonography was carried out underwater. The transducer was attached to the ventral surface of the body or put 1 cm away from the body. Transverse and sagittal sonograms were taken of the length of all gonads, and the sex was determined. Information on sex determination by ultrasonography is presented in

Anatomical study
In the anatomical study of the female fishes, gonads were cylindershaped and were soft and flexible. They were situated on two sides

F I G U R E 4 Schematic illustrations from female (a) and male (b)
Mugil cephalus. The topographic area of the gonad is shown in the illustration.
( Figures 1 and 2). The Left and right ovaries and testes were similar in length. The testis was longer and thinner than the ovary in each fish (Figures 2 and 3). Gonads were close together in the caudal part (Figures 1 and 2).

Ultrasonographic study
In the sagittal ultrasonograms of the female gonads, a cylinder struc-

DISCUSSION
In this study, form, size, echogenicity and, to some extent, the state of the gonad wall were appropriate indexes in identifying the male or female gonad. In all cases, the female gonad in ultrasonography had parenchyma which was completely homogenous, hypoechoic and a three-layer identifiable wall. In the case of the male gonad, the oval-shaped echogenic structure with a small diameter and a single layer was observed. M. cephalus is very sensitive to touching by hands, and more touching leads to the removal of the fish scales and even causes the death of the fish (Chang et al., 1999). Therefore, many techniques can be performed in other kinds of fish like operation and trocar; however, in these kinds of fish, those techniques cannot be used. Having the minimum touch with the body of the fish is one of the most important benefits of the ultrasonography technique in order to identify the sex compared to other techniques of sex identification like operation or trocar; besides the technique being aggressive, there is a lot of touching between the hand and the body of the fish. Even in sexual steroid evaluation techniques, it is necessary to catch the fish and to touch it (Chang et al., 1999). But in ultrasonography, the fish become still in the water with an anaesthetic, and the ultrasonography is carried out with a minimum touch. Even the transducer can be situated 2 cm away from the fish without anybody touching it (Karlsen & Holm, 1994). All these points enable identification with minimum touching of the fish.
Further, the speed of identifying sex in M. cephalus with ultrasonography is one of the important points, which makes the value of this technique more outstanding. As the needed time to perform sonography for A. stellatus was 30 s (Moghim et al., 2002), in this examination also the average needed time to identify the sex from the time the transducer is located on the body of the fish is less than 30 s. Another benefit that sex identification with ultrasonography has compared to sexual steroid evaluation methods is the needed time to announce the result of the diagnosis. In the sexual steroid evaluation methods, a long time is needed after taking the blood to test it and announce the result. During this period until the determination of the experiment test, the fish under the experiment must be kept separately or, after marking, in an individual pool. However, the result in ultrasonography is determined immediately, and the male and female fish are released separately in the pool-specific for male and female, and it saves time and cost (Moghim et al., 2002).
In the performed examination on the Atlantic salmon (Mattson, 1991), male and female gonads are flat and without lobule in observations, but in A. persicus (Vajhi et al., 1999) and A. stellatus (Moghim et al., 2002), the immature gonad stage III is as above and with lobulation in observation. The female gonad also has a heterogeneous state, and the edges are not flat. In M. cephalus, male and female gonads are observed early in their maturation, without lobule, and with flat edges. This issue is explained in Martin-Robichaud and Rommens (2001) in this way that the sexual glands of bony fishes are lobulated, but in some other fishes like Salmon and Mugilidae, they are flat and without lobule (Mattson, 1991).
The identification of immature gonads in the performed examinations upon the S. salar (Mattson, 1991) and G. morhua (Karlson & Holm, 1994) was difficult due to the small size of the gonad. Moreover, in the performed examination on A. persicus (Vajhi et al., 1999), A. stellatus (Moghim et al., 2002) and A. nudiventris (Vajhi et al., 2003), it is stated that the identification of sex in young and immature males is difficult due to the small gonad, and the most amount of error in sex identification is attributed to this group. Moreover, in this research, the sex identification of the immature female was difficult due to the small and narrow ovary. Using transverse ultrasonograms, to some extent, it was possible to identify the male gonad, but in sagittal ultrasonograms, it is As mentioned in the Introduction, the place to put the transducer on the body of the fish is of special importance. In our study, the place of the organs in the M. cephalus coelomic cavity and the obtained experience showed that for rapid and easy sex identification, the best place to put the transducer is on the ventral surface of the fish in the caudal part of the coelomic cavity for taking transverse and sagittal F I G U R E 8 Transverse ultrasonogram (left) and schematic illustration (right) of the testis (early in their maturation) of the Mugil cephalus from the ventral part of the body anterior to the pelvic fine ultrasonograms (between ventral and anal fins). Gonads are close to each other in the caudal part, so if the transducer is put more caudally, identification will be easier.  (Tung, 2021). According to the collected specimens from Caspian Sea (same place with our study), the maturation of L. aurata (another species of grey mullet found in the Caspian Sea) occurs at minimum age 3, and generally at ages 4-6. Similar results were reported by the Caspian Sea Biodiversity Database and some European regions (Fazli et al. 2008). Mattson (1991) mentioned the difference in ripe and immature material in S. salar is because of the smaller size of the ovary in immature fish, and this causes easier sex identification in mature materials.
Vajhi et al.'s study in 1999 on mature and immature A. persicus reported the sex identification in immature fish, especially the mature male, and the result is the small immature male gonad. Although our study is conducted early in their maturation fish, as there is an acceptable result, it appears in mature Mugilidae; it is also a good and more comfortable technique. There is a need for more study in mature Mugilidae; moreover, because of anatomic similarities of sexual glands in different species of Mugilidae, it appears that ultrasonography can be used to identify sex in other types of Mugilidae. Therefore, we can consider the ultrasonography technique in sex identification in Mugilidae (early in their maturation) an accurate, efficient, and rapid technique which is an appropriate alternative for the previously time-consuming and aggressive methods.

AUTHOR CONTRIBUTIONS
CRediT contribution not specified: